Discuss the challenges facing forensic scientists

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GENETICS FOR IDENTIFICATION ESSAY

Discuss the challenges facing forensic scientists for DNA-based identification of the remains of the victims of war or other conflicts (both civilian and military personnel).

Illustrate with a range of examples.

Discuss the challenges facing forensic scientists for DNA-based identification of the remains of the victims of war or other conflicts (both civilian and military personnel). Illustrate with a range of examples.

Introduction

The remains of victims of war often pose numerous challenges to forensic scientists enlisted to aid in the identification process using DNA analysis. Foremost, the remains of victims of war have frequently been buried for long periods of time, often over 50 years, and this can cause degradation and contamination of DNA. This affects the quality and quantity of extracted DNA, making it difficult to amplify and generate a genetic profile. This concise report highlights the problems facing forensic scientists during the analysis of war remains and the methods used to overcome some of these issues.

DNA techniques

The DNA techniques which can be used in DNA identification of skeletal bones include: mitochondrial DNA analysis (mtDNA); autosomal Short Tandem Repeat (STR) and Y-STR analysis; and mini-STR profiling. STRs, also known as microsatellites, are DNA repeat units between two and seven base pairs long that can easily be amplified by Polymerase Chain Reaction (PCR). The number of repeat units varies considerably amongst individuals, hence why sensitive STR analysis is highly discriminative for identification, even in degraded DNA samples (Butler et al., 2012).

Fifteen years ago, results of a study where teeth were buried in soil for up to eighteen weeks revealed that mtDNA analysis using primers for the HV1 and HV2 regions generated the best results in comparison to other DNA techniques; thus mtDNA was a reliable method for the identification of skeletal remains (Pfeiffer et al., 1999). Developments in technology mean that analysis of mtDNA alone is now insufficient information for positive identification, and more specific methods are employed, such as STR analysis. Nevertheless, when nuclear DNA (nDNA) samples are too degraded to be processed using STR analysis, identification using mtDNA and hypervariable regions is used. This is because mtDNA is present in high copy numbers and the circular structure of mtDNA makes it more resistant to degradation, but again there are limitations (Higgins et al., 2013; Coble et al., 2005). Mitochondrial DNA is passed on maternally; therefore it will be the same throughout generations of females of the same maternal lineage and could identify a familial match to a sister or aunt, for example. However reference samples are solely restricted to maternal relatives, thus its discriminatory power is far less than an STR match (Lee et al., 2010).

Finally, where female relatives are absent, Y-STR analysis can be carried out to identify paternal lineage of the Y-chromosome. Mutations within the Y-chromosome are possible, and they may occur between generations as well as within the same bone samples, particularly if they are old skeletal bones. This must be considered when making conclusions from Y-STR analysis as it may cause problems for the forensic scientist during the identification process (Borić et al., 2011).

DNA samples from skeletal remains

With war victim skeletal remains DNA sample options are minimal, but the preferential samples to obtain for DNA analysis are teeth and bones, which contain both nDNA and mtDNA. DNA in teeth is generally considered more protected against degradation and destruction than bones due their unique composition and their location in the jawbone protecting them against degrading exogenous organisms, making them useful for analysis decades after death. Additionally, they normally yield a higher quality of DNA than bones, and the results of a study by Pilli and colleagues looking into the effect of contamination on samples revealed that teeth have a greater refractory to contamination by exogenous DNA than bones (Higgins et al., 2013; Pilli et al., 2013 respectively). Another reason for utilising teeth is because there are a number of sources of DNA within the tooth, including dentine, and this dentine powder is rich in mtDNA (Muruganandhan et al., 2011).

Nevertheless, both of these samples are used because the internal content is unlikely to be contaminated with contemporary DNA compared to other samples, and the recovery of one or both of these is observed in a number of case studies identifying war victims (Lee et al., 2010; Marjanović et al., 2007; Andelinović et al., 2005; Ivanov et al., 1999). However, although these sources of DNA are the most likely to be preserved over time, DNA analysis can still be affected by the presence of PCR inhibitors (for example environmental and biological chemicals), insufficient quantity of DNA material, and high levels of DNA degradation (Marjanović et al., 2007).

DNA degradation

DNA degradation and PCR inhibitors occasionally cause allele and/or locus dropout, thus not reflecting the true profile of the individual and can cause problems for forensic scientists during the analysis of genotypic DNA profiles, especially if heterozygotes are interpreted as homozygotes (Coble et al., 2005). Exposure to environmental conditions affects DNA, and factors such as heat and humidity affect the rate of DNA degradation and the resulting quantity of DNA, with cooler temperatures delaying the degradation process. This DNA degradation caused by endogenous intracellular enzymes results in smaller DNA fragments sizes and may also cause base mutations (Higgins et al., 2013).

Despite the development of sensitive DNA typing identification methods, in some cases excessive DNA degradation can still pose an issue, as seen in Lee et al., 2010. One sample (SR0014) had an exceptionally low DNA yield for both extractions, 28.7±20.69 pg/µL and 27.2±16.81 pg/µL, in comparison to the average yield of 217.5 pg/µL and 199.1 pg/µL, respectively (table 1). This DNA yield produced genotypic results at eleven autosomal STR loci; however nine of these were homozygotic and this was interpreted as allelic dropout, which in turn causes problems for profile determination and does not allow for identity inclusion or exclusion. Another example of degraded DNA amplification being below 100% was seen using AmpFISTR Yfiler amplification, which revealed only 34 out of 49 profiles, and MiniFiler which produced 40 out of 49 genetic profiles, with the other profiles being incomplete, likely due to only small amounts of degraded DNA being amplified (Borić et al., 2011).

Table 1 – DNA concentrations (pg/µL) extracted from twenty-one skeletal samples belonging to Korean War victims. Two DNA samples were extracted from each bone sample and quantified to try and replicate the profile for consistency and to highlight any contamination issues. (See Lee et al., 2010. Available at: http://onlinelibrary.wiley.com/doi/10.1111/j.1556-4029.2010.01411.x/pdf. Last accessed on: 24th February 2014)

Number

Skeletal Sample

Concentration pg/µL (1st Extraction)

Concentration pg/µL (2nd Extraction)

1

SR0001

339.5 ± 09.02

160.6 ± 24.26

2

SR0002

213.5 ± 15.83

319.7 ± 53.33

3

SR0004

783.6 ± 20.51

361.4 ± 09.90

4

SR0008

613.6 ± 04.53

318.0 ± 14.76

5

SR0011

106.1 ± 05.49

82.3 ± 20.83

6

SR0012

84.3 ± 05.70

29.6 ± 03.11

7

SR0014

28.7 ± 20.69

27.2 ± 16.81

8

SR0015

119.0 ± 04.15

211.7 ± 11.20

9

SR0016

81.5 ± 10.46

60.2 ± 06.30

10

SR0017

70.6 ± 11.54

91.3 ± 30.38

11

SR0018

176.2 ± 06.73

266.6 ± 01.42

12

SR0078

161.8 ± 14.49

295.4 ± 14.60

13

SR0079

190.7 ± 31.71

224.5 ± 03.38

14

SR0080

169.2 ± 47.35

146.4 ± 04.40

15

SR0081

274.4 ± 38.36

305.1 ± 59.13

16

SR0083

51.2 ± 10.75

68.7 ± 18.25

17

SR0135

23.5 ± 05.20

73.4 ± 17.11

18

SR0221

303.1 ± 44.53

416.2 ± 48.97

19

SR0315

232.9 ± 01.28

129.7 ± 15.03

20

SR4003

57.3 ± 08.88

74.1 ± 05.64

21

SR7001

486.8 ± 03.15

518.5 ± 82.13

SUM

4567.5

4180.6

AVERAGE

217.5

199.1

DNA extraction

Another issue facing forensic scientists is the quantity of DNA extracted from bones or teeth. Degraded samples offer a paucity of template DNA concentration, hence the smaller PCR products (Ivanov et al., 1999). If precise procedures are not employed then DNA essential for producing a genotypic profile may be lost, and methods are required to maximise the potential from the extraction process. Recently, developments in such methods have massively impacted on the success of obtaining profiles from skeletal remains which are highly degraded. Extraction techniques employed by forensic scientists to overcome this challenge include the standard organic (phenol/chloroform) method, the PrepFiler Forensic DNA Extraction kit, the Qiagen DNA extraction procedure, and a large-scale silica based extraction method, with a minimum of two independent extractions for each sample usually taken. Lee and colleagues used the latter in 2010 alongside demineralisation to maximise the DNA yield, and the positive effect of this method was reflected in the high DNA yields observed following quantification, with all samples, except two, being greater than 50pg/L (Lee et al., 2010). Additionally, during the extraction of DNA from 109 bone samples from victims of war in mass graves in Croatia, an advanced extraction method, alongside the phenol/chloroform method, was used. The standard method yielded 20-100ng of extracted DNA across samples, whilst the advanced Promega DNA IQ system produced 20-200ng of DNA (Andelinović et al., 2005).

Contamination

The sensitivity of new DNA typing methodologies to minute amounts of DNA brings with it the challenge of contamination. Remains buried for a long period of time and then excavated for analysis are subject to natural cross contamination from foreign material from the surrounding environment as well as from human handling, and this can affect the validity of the findings and interfere with DNA profiles. Thus, procedures are implemented to remove any foreign matter from the outer surface which may contaminate the probe extracting the DNA from the core of the bone or tooth. Common protocols forensic scientists use to overcome the issue of contamination on bones include sanding down the outside of the bone, washing it in mild detergent, irradiating it with UV light and storing it at -20oC until it is required for DNA extraction (Ossowski et al., 2013; Borić et al., 2011; Lee et al., 2010; Marjanović et al., 2007; Imaizumi et al., 2002). However, in cases where excess contamination has occurred from archaeological handling and repeated excavation and reburial, it is impractical for forensic scientists to remove this extent of contamination, further limiting the samples which can be used for DNA.

It is of paramount importance that sterile environments are used for sample analysis and that gloves are worn when handling bones to avoid contamination from humans, and gloves should be changed between the handling of bones belonging to different individuals to avoid DNA transfer. Another issue regarding contamination is the processing of ancient skeletal samples in proximity to contemporary relative reference samples, therefore, DNA from these two sample types should be extracted and amplified in separate laboratories. The majority of studies referenced in this report have not stated whether they adhered to this, with the exception being Ossowski et al., 2013 who reported that all laboratory staff wore masks, lab overalls and sterile gloves and everyone who handled the samples had previously had their DNA sample taken for reference purposes. As a result of the strict procedures implemented, no contamination was seen throughout the examination process, and they successfully identified two individuals through DNA analysis when it was determined that they could not be identified through anthropological methods (Ossowski et al., 2013). Finally, a complete record of everyone that has handled the samples pre- and post-excavation should be kept (Pilli et al., 2013).

Issues with mass grave DNA samples

A further problem forensic scientists are confronted with is the high number and poor quality of the remains discovered, particularly in mass graves, due to wars being open events with large numbers of unknown individuals involved. The violent nature of wars and conflicts and circumstances of death often renders a number of remains fragmented, meaning that prior to DNA analysis, an anthropologist must examine and match bones suspected to belong to the same individual. This should be considered during DNA analysis in case different profiles arise from bones supposedly belonging to the same individual. Furthermore, explosives can cause bones to become incinerated, damaged and carbonised which will affect DNA extraction and amplification (Ivanov et al., 1999).

This issue was presented during the recovery of approximately 10,000 skeletal remains, belonging to approximately 53 war victims in 2009, more than sixty years after World War II. In the majority of cases, it was impossible to conclude which bones belonged to one individual due to the vast number of separated, damaged and intermingled bones and the small grave area to which they were confined. Additionally, a number of victims showed obvious gunshot wounds, which further shattered the skull bones into multiple fragments (Borić et al., 2011).

DNA reference samples

Prior to DNA analysis, it is paramount that circumstantial investigative research is carried out to narrow down and establish possible victims in the grave to allow for identification of their surviving relatives. This is problematic in the first instance if ante-mortem records of soldiers have not been kept. It is reasonable to assume that relatives of military personnel would be easier to identify, compared to civilians, because records are often kept of the soldiers that fought in a war, as well as when they were reported missing or pronounced dead. Reference swabs are required from these presumptive relatives to compare their DNA profiles with the profiles obtained from the unknown remains to determine if a familial match is seen.

Ivanov et al., 1999 reported absent DNA samples on record during their involvement in the identification of the remains recovered following the Chechen War (1994-1996). Few remains were available due to the lethal force of missiles, and this was an additional problem to the lack of DNA samples on record, which meant the absence of comparative reference samples. Accordingly, the timely process of locating potential relatives and collecting their DNA samples began. In comparison to some cases where excavated war remains are over 60 years old (Marjanović et al., 2007), these remains were excavated three years after the end of the war, however skeletonisation and advanced decomposition, with mummification, was still observed. Bones had been scavenged by animals, further exposing them to contamination alongside the reported careless excavation of the bodies (Ivanov et al., 1999).

Mini-STRs

Applying mini-STR loci to severely degraded DNA samples is effective in genotyping nDNA profiles that would otherwise yield a negative result with standard STR kits, which use STR loci of up to 250 base pairs and are likely to cause loss of signal (Andelinović et al., 2005). In comparison, mini-STR technology can amplify loci with alleles that have fewer than 150 base pairs and works by annealing primers as close as possible to the STR repeat region, creating the smallest possible amplicon (Figure 1) (Martin et al 2006; Pizzamiglio et al., 2006). When there is not enough intact DNA to produce full profiles using the larger CODIS loci, mini-STR loci markers are small enough to amplify alleles less than 150 bp in length (Hill et al., 2008). It is important to remember that mini-STRs are designed to profile high quantity, low quality, degraded DNA and should not be used for small amounts of DNA.

Fig. 1 – Mini-STR analysis uses primers which anneal as close as possible to the STR repeat region along the genome, creating a small amplicon to be amplified.

Successful application of mini-STRs was seen in 2010 when skeletal remains from the Korean War were subject to DNA analysis in the hope to identify the 55 year old remains of the missing casualties (Lee et al., 2010). Twenty-one skeletal samples were extracted from the remains of the victims of the Korean War and, following decontamination, were subjected to PCR amplification and sequencing of the mtDNA HV region, and PCR amplification of autosomal STRs and Y-STRs using common STR kits and in-house miniplex plus systems that use smaller amplicons to optimise the genetic material from degraded samples. Results revealed that mtDNA hypervariable regions were efficiently amplified and determined in all 21 samples. A combination of AmpFISTR Identifiler alongside size-reduced amplicons in AmpFISTR MiniFiler and the in-house miniplex NC01 plus system for autosomal STR, successfully genotyped 17.2 loci out of 18, and the miniplex NC01 system showed 100% success in genotyping the four loci due to the reduced amplicon size.

Additionally, twenty samples were successfully genotyped at 11 or more loci using standard STR kits, but with the in-house system, they were genotyped at 15 or more loci. The results of amplifying autosomal STRs showed the importance of mini-STRs when working with highly degraded DNA, as reduced-sized amplicons genotyped samples with a low quality of DNA in comparison to the standard kits. The same success of genotyping samples of poor quality DNA was seen using the Y-miniplex plus system in conjunction with AmpFISTR Yfiler during Y-STR genotyping (Lee et al., 2010).

Profiles were also generated using mtDNA PCR amplification and miniplex NC01 analysis using the buccal samples provided by the 24 suspected relatives (Lee et al., 2010). Smaller quantities of template DNA were extracted from the relatives, as although more DNA was available, it was of a higher quality compared to the degraded unknown samples, so a smaller amount was required. If too much high quality template DNA is used, excess peaks and spurious bands would be observed, making the results unclear (Ivanov et al., 1999), and alternatively too little, or degraded DNA, may reduce the height of some alleles so the peak heights may be too low to distinguish from background noise (NFSTC Science Serving Justice, 2007).

Conclusion

In conclusion, it is evident that DNA degradation affecting the quality and quantity of DNA, fragmentation of bones, numbers of bones in mass graves, contamination and poor extraction procedures are all challenges that forensic scientists face during DNA based identification of skeletal war victim remains. However, as efficient DNA extraction methods to optimise the template DNA concentration are advancing, and technology is consistently being refined to develop methods such as the mini-STR system, identification of war victims using DNA analysis, alongside anthropological measures, is becoming more successful.

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